Studies on the active site of bovine liver argininosuccinase will be continued. The results of covalent modification of the enzyme with a 14C bromo analogue of fumerate are in agreement with the stoichiometry expected for the number of active sites. This has formed the basis of the procedures planned for isolation of the radioactive peptide fragment representing the region of the active site. It is also planned to undertake identification of the modified amino acid in the peptide fragment. To facilitate the latter, alkylated derivatives of the several most likely amino acid candidates will be synthesized as models. The large-scale purification of bovine brain argininosuccinase has been greatly advanced in the past year and is estimated to be close to homogeneity. With the homogeneous enzyme it is planned to carry out a detailed comparison of physical and kinetic properties with those of the kidney and liver enzyme in so far as the limited amount of highly purified brain enzyme permits. The details of a radioassay for argininosuccinase have been developed with 14C-labeled argininosuccinate to avoid troublesome colorimetric procedures. The method depends on reaction product separation by fractional ion exchange chromatography. As all procedural details have been checked at all stages by an independent assay, it is now planned to apply the method to crude homogenates of rat liver and other rat tissues and to increase the sensitivity further to permit assay of cells grown in culture. BIBLIOGRAPHIC REFERENCES: Preparation of N-alpha-Acetyl-L-Ornithine from N-alpha-Acetyl-L-Arginine, a New Substrate for Arginase. S. Ratner, Analytical Biochemistry, 73, 423-429 (1976). Enzymes of Arginine and Urea Synthesis, Sarah Ratner, in The Urea Cycle, S. Grisolia, Ed., J. Wiley, New York, 1976, pp. 181-219.